The present invention is related to a method for isolating hepatitis B surface antigen (or "HBsAg") from transformed yeast cells.
It is known that hepatitis B surface antigen protein can be purified from infected human plasma and used as a vaccine to prevent hepatitis B virus infection. HBsAg genes have been cloned and expressed in E. coli, yeast cells and mammalian cells to reduce the dependency on plasma. The synthesis of HBsAg recovered as 22 nm particles in yeast cells, e.g. Saccharomyces cerevisiae, has been reported by Valenzuela et al., Nature, 298:347-350 (1982); by Hitzeman et al., Nucleic Acid Research, 11:2745-2763 (1983) and by Miyanohara et al., Proc. Nat'l Acad. Sci., 80:1-5 (1983).
Recombinant HBsAg protein particles produced by transformed Saccharomyces cerevisiae (Baker's yeast) have been isolated as described in U.S. Pat. No. 4,707,542. Normally, the expressed HBsAg was separated and purified from cell homogenate via the addition of protease inhibitor of phenylmethylsulfonylfluoride (PMSF) and non-ionic detergent polyoxyethylene (9,5)-p-t-octyl-phenol (Triton.RTM. X-100) to the cell homogenate. Then, chaotropic agents such as potassium or sodium thiocyanate (KSCN or NaSCN) or urea, can be used to formulate different forms of surface antigen protein aggregates followed by adsorption onto fused silica, borate N-morpholine propanesulfonic acid (MOPS) buffer elution, hydrophobic interaction chromatography on butyl agarose, treatment with polystyrene beads cross-linked with divinylbenzene (Amberlite XAD-2 resin), ultrafiltration, sepharose 6B chromatography, as well as immunoaffinity and ECTHAM-cellulose chromatography purification steps.
An improvement on the hydrophobic interaction chromatography using various hydrophobic groups as the fixing carrier was reported in European Patent publication No. 0179485A2. Also, U.S. Pat. No. 4,624,918 described a method for the purification of HBsAg from recombinant cell culture via concentration, precipitation, immunoaffinity column, anion exchange and gel permeation chromatography.